Deacetylnemorone Abietane Diterpenoids for Use in Cancer Treatment

ABSTRACT

The deacetylnemorone shows efficacy in treatment and prevention of a wide range of cancer types as well as other neoplastic diseases as a chemo-preventative, a primary or secondary cytotoxic agent, a sensitizer for other therapies, or one component of a combinatorial treatment.

CROSS REFERENCE TO RELATED APPLICATION

This application claims filing benefit of U.S. Provisional PatentApplication Ser. No. 62/518,051 entitled “Use of a Newly DiscoveredSesterterpenoid for Cancer Treatment” having a filing date of Jun. 12,2017, and U.S. Provisional Patent Application Ser. No. 62/628,416entitled “Deacetylnemorone Abietane Diterpenoids for Use in CancerTreatment” having a filing date of Feb. 9, 2018, both of which areincorporated herein by reference for all purposes.

FEDERAL RESEARCH STATEMENT

This invention was made with Government support under Grant No. 1631439,awarded by the National Science Foundation. The Government has certainrights in the invention.

BACKGROUND

Cancers remain the cause of poor health and early death throughout theworld. Despite advances in treatment, cancer remains the second leadingcause of death in the United States and is the leading cause of death in21 states as of 2016. Further, the probability of being diagnosed withan invasive cancer was found to be 42% for men and 37.6% for womenliving in the United States according to a study by Siegel and others(“Cancer statistics, 2016,” CA Cancer J Clin, vol. 66, no. 1, pp. 7-30,2016 January-February 2016). The same study reported that 582,623 deathsin 2012 were a result of cancer and projected that 1,685,210 new cancercases and 595,690 cancer deaths would occur during 2016.

Treatment of cancers has traditionally been accomplished through one of,or a combination of, chemotherapy, surgery, radiotherapy, immunotherapy,and hormone therapy, among others. Unfortunately, differences in geneticexpression, drug sensitivity, cell morphology, and metastatic targetsacross the dozens of known cancer types has stymied the long-termsuccess of treatments. Acquired drug resistance in response tochemotherapy remains another hurdle in cancer treatment that demands adiverse arsenal of cytotoxic agents. Cancer recurrence and distantmetastases, potentially explained by inherently robust and drugresistant cancer stem cells, further hinder positive prognoses in cancerpatients. It is hypothesized that cancer stem cells can remain dormantand undetected in the body for years before reactivating and beginningthe formation of a new tumor. As such, cancer recurrence and distantmetastases continue to plague cancer patients after months or even yearsof remission using current treatments.

The role of immune cells in combating aggressive tumors has becomeincreasingly recognized in the medical community and has led to newapproaches for cancer therapies. FDA approval of the first therapeuticcancer vaccine, sipuleucel-T, and other cancer immunotherapy drugs,including monoclonal antibodies such as ipilimumab, in addition toincreased understanding of the immune system's role in the tumormicroenvironment, has led to a call for small molecules capable ofregulating immune activity and supporting tumor death.

In spite of such advances, many shortcomings and disadvantages ofcurrent chemotherapeutics and other cancer treatments are readilyapparent. The hair loss, nausea, vomiting, loss of appetite, compromisedimmune system, and other side effects commonly associated with a cancertherapy are often a consequence of the currently utilized treatments andnot the disease itself.

Unfortunately, a push to develop chemotherapeutic drugs capable oftargeting a specific molecule or cancer-associated signaling pathwaywith reduction in side effects has failed to yield the expectedimprovements in patient prognoses. This is largely due to the ability ofcancer cells to utilize a combination of many different cellularmechanisms to enhance viability. In many cases, cancer cells are able tocircumvent apoptosis induced from targeted therapies by simplyactivating other survival pathways after the initial treatment.

Natural products are a historically successful source of medicinallyactive compounds with fewer unwanted side effects, especially in regardto chemotherapeutics. In fact, 63% of cancer drugs used between 1981 and2006 were natural products, were inspired by natural products, or weresynthesized from a natural pharmacophore. Medicinally active compoundsderived from natural materials have the potential to provide targetedcytotoxic and immune modulating responses while limiting the taxing sideeffects associated with currently utilized cancer treatments. The use ofnatural products attempts to balance a robust ability to target numerouspathways simultaneously with a historical record of safe humanconsumption and benign side effects.

There is a need to discover and optimize the use of novel cytotoxiccompounds with low IC₅₀ values, diverse biological targets, immuneregulatory capability, and diminished side effects. In addition, thereis a need for new treatments engineered to target pathogenic cells bothduring initial treatment of cancers and after remission has occurred inorder to prevent cancer recurrence. New treatments based upon naturalmaterials that can provide efficacy with benign or limited side effectswould be of great benefit.

SUMMARY

According to one embodiment, disclosed is a method for inhibiting thegrowth and development of cancer cells. The method includes delivering adeacetylnemorone abietane diterpenoid to an area comprising cancercells. Beneficially, the method shows efficacy for a large variety ofdifferent cancer cell types.

Also disclosed is a method for preventing angiogenesis, for instance inthe treatment of cancer. The method includes delivering adeacetylnemorone abietane diterpenoid to an area comprising endothelialcells.

Also disclosed is a composition configured for inhibiting the growth anddevelopment of cancer cells and/or inhibiting angiogenesis that includesa deacetylnemorone abietane diterpenoid and a pharmaceuticallyacceptable carrier. The deacetylnemorone can have the followingstructure:

or a tautomer there of in which R₁, R₂, and R₃ are independentlyselected from —H, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ alkenyl, C₁₋₁₀alkenoxy, —OH, —OAc, —CHO, -Ph, —OC₆H₅, —OC₆H₄OH, —COC₆H₅, —OCONH₂,—OCONHCH₃, —OCOC₆H₄NH₂, —NH₂, or ═O.

BRIEF DESCRIPTION OF THE FIGURES

A full and enabling disclosure of the present subject matter, includingthe best mode thereof to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures in which:

FIG. 1 illustrates the structure of one embodiment of a deacetylnemoroneabietane diterpenoid encompassed herein.

FIG. 2 illustrates the Time of Flight mass spectometry (TOF-MS) spectrum(negative ion mode) of the deacetylnemorone of FIG. 1.

FIG. 3 illustrates the high resolution mass spectometry (HR-MS) spectrum(negative mode) of the deacetylnemorone of FIG. 1.

FIG. 4 illustrates the hydrogen nuclear magnetic resonance (¹H-NMR)spectrum of the deacetylnemorone of FIG. 1.

FIG. 5 illustrates the carbon 13 nuclear magnetic resonance (¹³C-NMR)spectrum of the deacetylnemorone of FIG. 1.

FIG. 6 illustrates the hydrogen correlated spectroscopy (H—H COSY)spectrum of the deacetylnemorone of FIG. 1.

FIG. 7 illustrates the heteronuclear single quantum coherence (HSQC)spectrum of the deacetylnemorone of FIG. 1.

FIG. 8 illustrates the heteronuclear multiple bond correlation (HMBC)spectrum of the deacetylnemorone of FIG. 1 (in DMSO-d6).

FIG. 9 presents the percent viability of MG-63 cancer cells in responseto various concentrations of a deacetylnemorone after 48 and 72 hours ofexposure.

FIG. 10 presents the percent viability of SK-OV-3 cancer cells inresponse to various concentrations of a deacetylnemorone after 48 and 72hours of exposure.

FIG. 11 presents the percent viability of MDA-MB-231 cancer cells inresponse to various concentrations of a deacetylnemorone after 48 and 72hours of exposure.

FIG. 12 presents the percent viability of HCT-116 cancer cells inresponse to various concentrations of a deacetylnemorone after 48 and 72hours of exposure.

FIG. 13 presents the percent viability of HCT 116/200 cancer cells inresponse to various concentrations of a deacetylnemorone after 48 and 72hours of exposure.

FIG. 14 presents the percent viability of A2789ADR cancer cells inresponse to various concentrations of a deacetylnemorone after 48 and 72hours of exposure.

FIG. 15 presents a waterfall plot of the growth percent of 59 cell linesin response to 10 μM of a deacetylnemorone as determined by a NCI-60one-dose screen.

FIG. 16 presents the percent viability of HCT 116/200 cancer cells inresponse to a 48 hour exposure of a deacetylnemorone alone and inconjunction with 5-fluoro-2′-deoxyuridine (FdUrd).

FIG. 17 presents the cell number of HCT 116/200 cancer cells in responseto a 48 hour exposure of a deacetylnemorone alone and in conjunctionwith 5-fluoro-2′-deoxyuridine (FdUrd).

FIG. 18 presents a histogram of PI expression as measured by flowcytometry for SKMEL5 cells treated with control or 15 μM of adeacetylnemorone.

FIG. 19A presents cell cycle flow cytometry data showing the percent ofanalyzed SKMEL5 cells in each phase of the cell cycle after fourdifferent time points of treatment with control.

FIG. 19B presents cell cycle flow cytometry data showing the percent ofanalyzed SKMEL5 cells in each phase of the cell cycle after fourdifferent time points of treatment with 15 μM of a diacetylnemorone.

FIG. 20 presents the results of an invasion assay of MDA-MB-231 breastcancer cells when incubated with 1 or 10 μg/mL of a deacetylnemorone 6,20, and 26 hours. (Significant difference from the control of the sametime point was denoted by * p≤0.05, ** p≤0.01).

FIG. 21 presents representative images of the cells of FIGS. 20 at 0 and26 hours after insert removal and compound addition.

FIG. 22 presents the percent viability of HUVEC endothelial cells inresponse to 48 hours of incubation with various concentrations of adeacetylnemorone. The DOX control was incubated with 1 μg/mL DOX.

FIG. 23 presents the average number of junctions, or tubes, formedbetween HUVEC endothelial cells after 8 hours of incubation on growthfactor reduced BD Matrigel with growth media alone and with variousconcentrations of a deacetylnemorone.

FIG. 24A is a representative bright field image of the incubation ofHUVEC endothelial cells on growth factor reduced BD Matrigel in growthmedia alone.

FIG. 24B is a representative bright field image of the incubation ofHUVEC endothelial cells on growth factor reduced BD Matrigel in growthmedia including 0.1 μg/mL deacetylnemorone.

FIG. 24C is a representative bright field image of the incubation ofHUVEC endothelial cells on growth factor reduced BD Matrigel in growthmedia including 1 μg/mL deacetylnemorone.

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments of thedisclosed subject matter, one or more examples of which are set forthbelow. Each embodiment is provided by way of explanation of the subjectmatter, not limitation thereof. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present disclosure without departing from the scope or spirit ofthe subject matter. For instance, features illustrated or described aspart of one embodiment, may be used in another embodiment to yield astill further embodiment.

The present disclosure is generally directed to the utilization of anabietane diterpenoid that has been found to exhibit efficacy against alarge variety of cancer cells and that has also been found to preventangiogenesis. More specifically, disclosed are methods for inhibitingthe growth and development of pathogenic cells, and in one particularembodiment of cancer cells, by use of a deacetylnemorone abietanediterpenoid. The deacetylnemorone abietane diterpenoids encompassedherein can generally have a structure as follows:

or a tautomer there of in which R₁, R₂, and R₃ are independentlyselected from —H, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ alkenyl, C₁₋₁₀alkenoxy, —OH, —OAc, —CHO, -Ph, —OC₆H₅, —OC₆H₄OH, —COC₆H₅, —OCONH₂,—OCONHCH₃, —OCOC₆H₄NH₂, —NH₂, or ═O. In one embodiment, adeacetylnemorone abietane diterpenoid can have a structure asillustrated in FIG. 1.

The deacetylnemorone of FIG. 1 is an abietane diterpenoid that has beenisolated from the plant Salvia leriifolia. Salvia leriifolia Benth.(vernacular names include Nuruozak and Jobleh) is a perennial herbaceousplant that grows exclusively in south and tropical regions of certainareas of Iran. Unlike other species of the Salvia genus, the chemicalconstituents of S. leriifolia are not well recognized. The stem oil ofthe plant is known to include monoterpenes and sesquiterpenes, and inleaf and flower oils monoterpenes predominate over sesquiterpenes. Inrecent years, properties of this plant such as the attenuation ofmorphine dependence, hypoglycemic, antinociceptive and antiinflammatory,antioxidant, anti-ischemia, anticonvulsant, antiulcer effects,antibacterial activities and antimutagenic effects have been evaluated(see, e.g., Hosseinzadeh, et al. Iranian Journal of Basic MedicalSciences, 12:1, 2009, 1-8). However, the abietane diterpenoid of FIG. 1has not been previously isolated from the plant and the efficacy of thiscompound isolated from the S. leriifolia as well as derivatives asdescribed herein in inhibition of pathogenic cells as well as ininhibition of angiogenesis has not been previously recognized.

Deacetylnemorone as illustrated in FIG. 1 was isolated from S.leriifolia and purified before being exposed, in vitro, to immortalizedtumor cells. Deacetylnemorone of S. leriifolia as well as derivativesand tautomers as encompassed herein can display efficacy against a broadspectrum of pathogenic cells involved in multiple different types ofcancers and other neoplastic disorders. Beneficially, the describeddeacetylnemorones can show efficacy as a chemo-preventative, a primarycytotoxic agent, a sensitizer for other therapies, one component of acombinatorial anti-cancer treatment, or an anti-angiogenic agent.

As an effective inhibitor of angiogenesis, deacetylnemorone abietanediterpenoids as described can be a suitable therapeutic alternative fortreatment of disorders in which excessive blood vessel growthcontributes to the pathology, including cancer, rheumatoid arthritis,and psoriasis. For example, a deacetylnemorone may be suited fortreatment of atherosclerosis due to its anti-angiogenic properties.Inhibiting angiogenesis may prevent intraplaque angiogenesis, therebylimiting plaque growth, destabilization, and subsequent rupture.

Deacetylnemorone abietane diterpenoids as encompassed herein can exhibitanti-proliferative or cytotoxic effects in vitro after about 48 hours ofexposure (e.g., about 48 hr. to about 72 hr. of exposure) and have shownefficacy against a number of immortalized cancer cell lines. Forexample, disclosed materials and methods can be utilized to inhibitgrowth and development of cancer cells including, but without limitationto, osteosarcoma cells, ovarian adenocarcinoma cells, breastadenocarcinoma cells, colorectal carcinoma cells, as well as pathogeniccells present in angiogenic disorders. Beneficially, thedeacetylnemorones can exhibit efficacy against cancer cells exhibitingresistance to other, more traditional chemotherapies such as FdUrdutilized in treatment of colorectal cancer and doxorubicin utilized intreatment of a wide variety of cancers. For example, deacetylnemoroneabietane diterpenoids as described can inhibit growth and/or developmentof pathogenic cells including, without limitation, breast cancer cells,bladder cancer cells, Kaposi's sarcoma cells, lymphoma cells, ovariancancer cells, prostate cancer cells, central nervous system (CNS) cancercells, renal cancer cells, melanoma cells, colon cancer cells, non-smallcell lung cancer cells, and leukemia cells. The disclosed methods andmaterials can be particularly beneficial against cells that areuntreatable through current methods or that have developed a resistanceto other, more traditional treatments.

The materials and methods can be targeted in one embodiment againstcancer stem cells, which can aid in prevention of cancer metastasis andrecurrence.

In one embodiment, disclosed materials can be utilized to target cancertumors, and can do so without affecting healthy cells, leading todevelopment of treatment protocols having fewer side effects.

As described further in the Examples section below, in vitro toxicityscreening has demonstrated a synergistic relationship between thedisclosed materials and the commonly used chemotherapy drug FdUrd ininducing cell death in colorectal carcinoma cells. As such, disclosedmaterials can be utilized in one particular embodiment in combinationwith more traditional chemotherapy and in particular with moretraditional colorectal chemotherapy such as FdUrd. Further, disclosedmaterials can act synergistically with other treatments in clinical useor clinical trials including, but not limited to, surgical resection oftumors, radiation therapy, hormone therapy, immunotherapy, cancervaccines, etc.

The clinical application and dosage of disclosed materials can betailored to the particular cancer cell of interest as well as to tumorsize and stage, patient size, patient medical history, method ofdelivery, etc., according to methods as are generally known in the art.Use of these compounds in combination with any number of other bioactiveagents, e.g., chemotherapy agents such as FdUrd, has the potential toelicit desirable responses in a large variety of cancer cell types.

By way of example and without limitation, one or more deacetylnemoronesas described herein can be utilized in combination with other bioactiveagents including classes of antineoplastic drugs includingalkaloids/natural products, alkylating agents, antibiotics,antimetabolites, enzymes, farnesyl transferase inhibitors,immunomodulators, immunotoxins, monoclonal antibodies, oligonucleotides,platinum complexes, retinoids, tyrosine kinase inhibitors, androgens,antiadrenals, antiandrogens, antiestrogens, antiprogestins, aromataseinhibitors, estrogens, LH-RH analogs, progestogens, and somatostatinanalogs. Examples of common chemotherapeutics on the market that can beutilized in conjunction with deacetylnemorone can include, withoutlimitation, the alkaloids paclitaxel, vinblastine, and vincristine; theantimetabolites gemcitabine, 5-fluoruracil, and methotrexate; theantibiotic or antibiotic derivatives doxorubicin, daunorubicin, andbleomycin; the hormonal antineoplastics tamoxifen, diethylstilbestrol,and polyestradiol phosphate; and the immonomodulators sipuleucel-T,interferon-5, and nivolumab.

When utilized in conjunction with another therapy, disclosed materialscan be administered at the same time as the other therapy, e.g.,together in a single composition, or at a different time or on adifferent schedule, e.g., prior to and/or following administration of asecond bioactive agent.

The methods can be utilized in vivo for treatment of cancer or in vitrofor study of pathogenic cells or tissue. In order for disclosedmaterials to be effectively utilized in a clinical therapy, adeacetylnemorone can be delivered so as to be provided with suitablebioavailability. According to one treatment method, a compositionincluding a deacetylnemorone and a pharmaceutically compatible carriercan be delivered to targeted cells via any pharmaceutically acceptabledelivery system. In general, a deacetylnemorone may be administered to asubject according to known methods, such as intravenous administrationas a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. Osmotic mini-pumps may also be used to providecontrolled delivery of a deacetylnemorone through cannulae to the siteof interest, such as directly into a metastatic growth. In certainembodiments, a deacetylnemorone can be administered directly to the areaof a tumor or cancer tissue, including administration directly to thetumor stroma during invasive procedures. A deacetylnemorone may also beplaced on a solid support such as a sponge or gauze for administration.

Pharmaceutically acceptable carriers include, but are not limited to,saline, buffered saline, glucose in saline, etc. Solid supports,liposomes, nanoparticles, microparticles, nanospheres or microspheresmay also be used as carriers for administration of deacetylnemorone. Asused herein the term “pharmaceutically acceptable carrier” is intendedto include any and all solvents, solubilizers, fillers, stabilizers,binders, absorbents, bases, buffering agents, lubricants, controlledrelease vehicles, diluents, emulsifying agents, humectants, lubricants,dispersion media, coatings, antibacterial or antifungal agents, isotonicand absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well-known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary agents can also be incorporated into the compositions.

The appropriate dosage (“therapeutically effective amount”) of thedeacetylnemorone can depend, for example, on the severity and course ofthe cancer, whether the deacetylnemorone is administered for therapeuticpurposes or in prevention of side effects of a chemotherapy, previoustherapy, the patient's clinical history and response to thedeacetylnemorone, and the discretion of the attending physician, amongother factors. A deacetylnemorone can be administered to a subject atone time or over a series of treatments and may be administered to thesubject at any time.

In one embodiment, a therapeutically effective amount of adeacetylnemorone can be in the range of about 0.001 mg/kg bodyweight/day to about 100 mg/kg body weight/day whether by one or moreadministrations for instance at a concentration of from about 1 mg/mL toabout 50 mg/mL. For example, a deacetylnemorone can be administered inan amount of from about 1 mg/kg body weight per day to about 50 mg/kgbody weight/day, in some embodiments. For instance, the deacetylnemoronecan be provided to the targeted site, e.g., a tumor or an in vitrodeposit of cancer cells, such that the deacetylnemorone is at aconcentration of about 10 millimolar (10 mM) or greater at the site ofcontact, for instance at a concentration of from about 10 mM to about 50mM in some embodiments. As expected, the dosage can be dependent on thecondition, size, age and condition of the patient.

A deacetylnemorone may be administered, as appropriate or indicated, ina single dose as a bolus or by continuous infusion, or as multiple dosesby bolus or by continuous infusion. Multiple doses may be administered,for example, multiple times per day, once daily, multiple times perweek, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeksor monthly. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques.

It can be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein includes physically discrete unitssuited as unitary dosages for the subject to be treated; each unit maycontain a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe application is dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Pharmaceutical compositions for parenteral, intradermal, or subcutaneousinjection can include pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycoland the like), carboxymethylcellulose and suitable mixtures thereof,vegetable oils (e.g., olive oil) and injectable organic esters such asethyl oleate. A composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like that can enhance the effectiveness of the activeingredient. Proper fluidity may be maintained, for example, by the useof coating materials such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. A composition may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.It may also be desirable to include isotonic agents such as sugars,sodium chloride and the like.

For intravenous administration, suitable carriers include, withoutlimitation, physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, an injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, aluminum monostearate or gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of an orally ingestible composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils may be added. Aliquid form may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, a composition can contain from about 0.5 to 90% by weightdeacetylnemorone, in one embodiment from about 1 to 50% by weightdeacetylnemorone.

For administration by inhalation, the deacetylnemorone can be deliveredin the form of an aerosol spray from pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, a pharmaceutical composition can be formulatedfor sustained or controlled release of deacetylnemorone. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially. Liposomal suspensions (including liposomes targeted toinfected cells with monoclonal antibodies to viral antigens) can also beused as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art.

It is to be understood that the in vivo methods have application forboth human and veterinary use. The methods of the present inventioncontemplate single as well as multiple administrations, given eithersimultaneously or over an extended period of time.

The present disclosure may be better understood with reference to theExample set forth below.

Example 1

The structure of a natural isolated deacetylnemorone (FIG. 1) wasestablished by means of 1D- and 2D-Nuclear Magnetic Resonance (NMR)(COSY (results shown in FIG. 6), HSQC (FIG. 7), and HMBC (FIG. 8)) andthe molecular formula was determined by High Resolution MassSpectrometry (HR-MS) (FIG. 3) and low resolution Time of Flight MassSpectrometry (TOF-MS) (FIG. 2). NMR spectra (FIG. 4 and FIG. 5) wereobtained in DMSO-d6.

The deacetylnemorone was found to have a molecular formula as shown inFIG. 1 of C₂₀H₂₆O₅ (evidenced by HR-MS ([M-H]⁻ ion at m/z 345.1707; FIG.3)). The NMR spectra (FIG. 4, FIG. 5) confirmed molecular formula ofC₂₂H₃₀O5. Resonance for two tertiary methyls (δH 0.67 (s, H3-18) and0.90 (s, H3-19)), two secondary methyls (δH 1.17 (d, H3-16) and 1.13 (d,H3-17)), and one oxygenated methine (δH 4.70 9(t, H-7)) were observed in¹H NMR spectrum. Complementary assignment was obtained through analysisof 2D spectra. The NMR spectra confirmed the abietane diterpenoidstructure.

Example 2 Materials and Methods Cell Culture

The human cell lines used for MTS assay screening included MG-63(osteosarcoma), SK-OV-3 (ovarian adenocarcinoma), MDA-MB-231 (breastadenocarcinoma derived from a metastatic site), HCT 116 (colorectalcarcinoma), HCT 116/200 (an FdUrd resistant subclone of HCT 116 cells),A2780ADR (a doxorubicin resistant subclone of the ovarian carcinomaA2780), and HUVEC (normal human umbilical vein endothelial cells). Allcells were stored in liquid nitrogen until use. MG-63, SK-OV-3,MDA-MB-231, and HCT 116 cell lines were all obtained from ATCC. A2780ADRcells were obtained from Sigma-Aldrich. HCT 116/200 cells were obtainedfrom Dr. Franklin G. Berger from the Center for Colon Cancer Research.HUVEC cells were obtained from Lonza. MG-63 cells were maintained in MEMmedium (Corning) supplemented with 10% Fetal Bovine Essence (VWR) and 1%penicillin/streptomycin solution (Corning). SK-OV-3 cells weremaintained in McCoy's 5A Medium (Sigma) supplemented with Fetal BovineEssence and 1% penicillin/streptomycin. A2780ADR cells were maintainedin RPMI 1640 medium (Corning) supplemented with 10% Fetal Bovine Essenceand 2 mM L-glutamine (ThermoFisher). MDA-MB-231, HCT 116, and HCT116/200 cells were all maintained in RPMI 1640 medium supplemented with10% Fetal Bovine Essence and 1% penicillin/streptomycin. HUVEC cellswere maintained in EGM-2 medium (Lonza BulletKit). Cells were incubatedat 37° C. and 5% CO₂ throughout the experiment.

MTS Assay

Cells were first grown to approximately 80% confluency within 25 cm³tissue culture flasks (VWR). The cells were washed once with PBS(Corning) before being trypsinized using a 0.25% trypsin, 2.21 mM EDTA,and sodium bicarbonate solution (Corning). Trypsinization was arrestedthrough addition of culture media, and the cell mixtures werecentrifuged at 2500 rpm for 5 minutes. Cells were counted using ahemocytometer and their viability was confirmed using trypan blue(Gibco). Cells were then seeded into 96 well tissue culture plates(VWR). MG-63, SK-OV-3, and A2780ADR cells were seeded at a density of2,000 cells/well with a total volume of 100 μL in each well. MDA-MB-231cells were seeded at a density of 5,000 cells/well with a total volumeof 100 μL in each well. HCT 116 and HCT 116/200 cells were seeded at adensity of 4,000 cells/well with a total volume of 100 μL in each well.HUVEC cells were seeded at a density of 3,000 cells/well with a totalvolume of 100 μL in each well.

After seeding, the cells were incubated for 24 hours at 37° C. and 5%CO₂ to allow for cell attachment and renewal of the exponential growthphase. The media was then removed and replaced with media supplementedwith the desired concentration of the deacetylnemorone of FIG. 1. Thedeacetylnemorone was stored at 4° C. until being first dissolved at10000 μg/mL in DMSO and subsequently diluted in culture media to thedesired concentration. The vehicle control was cell culture mediumsupplemented with 0.5% DMSO (Macron Fine Chemicals), representing thehighest final concentration of DMSO used to dissolve thedeacetylnemorone. For comparison, doxorubicin hydrochloride, or DOX(Sigma), and FdUrd (Sigma) supplemented treatments at concentrationssufficient to induce cell death in a majority of cells were alsoperformed. In combination studies, the deacetylnemorone and FdUrd werefirst mixed into the same media and then added to the cells. At either48 or 72 hours, the media was again removed, the cells were washed withPBS, and media containing 20% MTS solution (Promega) was added to thecells. The cells were incubated for 2 hours, and the absorbance of eachwell at 490 nm was measured using a Spectramax 190 microplate reader.

NCI60 Cytotoxicity Screening

One dose cytotoxicity screening was performed by the NCI-60 screeningprogram of the National Institutes of Health (NIH), which is known inthe art and available from the NIH. The NCI-60 panel is a collection ofcancer cell lines, from various diverse tumors, which was developed inthe 1980s by the National Cancer Institute to aid screening efforts forcytotoxic or cytostatic compounds.

In these screens 59 cell lines were utilized to determine the cytotoxiceffect of the deacetylnemorone. The cell lines included: ovarian cancercell lines OVCAR-8, IGROVI, NCI/ADR-RES, OVCAR-5, SK-OV-3, OVCAR-3,OVCAR-4; breast cancer cell lines HS578T, MDA-MB-231/ATCC, MCF7,MDA-MB-468, T-47D, BT-549; prostate cancer cell lines DU-145, PC-3; CNScancer cell lines U251, SNB-19, SF-539, SF-268, SNB-75; SF-295; renalcancer cell lines TK-10, SN12C, CAKI-1, ACHN, UO-31, RXF 393, A498,786-0; melanoma cell lines MDA-MB-435, UACC-257, LOX IMVI, SK-MEL-28,M14, SK-MEL-2, MALME-3M, UACC-62, SK-MEL-5; colon cancer cell linesSW-620, KM12, HT29, HCT-15, HCC-2998, COLO 205, HCT-116; non-small celllung cancer cell lines AS49/ATCC, NCI-H23, HOP-62, NCI-H226, NCI-H322M,NCI-H460, EKVX, HOP-92, NCI-H522; and leukemia cell lines K-562, SR,CCRF-CEM, MOLT-4, HL-60(TB), RPMI-8226.

For the assay, each cell line was treated with 10 μM of deacetylnemoroneto determine cytotoxicity. Each tumor cell line was maintained in RPMI1640 media supplemented with 5% fetal bovine serum and 2 mM L-glutamine.The cells were then seeded into 96 well plates at a cell density rangingfrom 5,000 to 40,000 cells per well depending on the cell line (eachwell containing 100 μL of cell suspension). The cells were thenincubated for 24 hours at 37° C., 5% CO₂, and 100% relative humidity toallow the cells to attach (if they were attached cell lines) and resumetheir growth phase. Two plates of each cell line which had not beenexposed to deacetylnemorone were then fixed with TCA at the time whenthe deacetylnemorone was added to the remaining plates. The remainingplates were treated with 100 μL of a dilution of deacetylnemorone whichhad been made by diluting the compound in DMSO to 400 times theexperimental concentration and subsequently diluting the solution inculture media supplemented with 50 μg/mL gentamycin to twice theexperimental concentration.

A control for each cell type was also simultaneously created, which wastreated with 100 μL of culture media without deacetylnemorone. The cellswere then incubated for 48 hours at 37° C., 5% CO₂, and 100% relativehumidity. In the case of the attached cell lines, the cells were thenfixed in situ by the gentle addition of cold TCA (10% final TCAconcentration) and incubated for 1 hour at 4° C. The plates were washedwith tap water five times and air dried. The fixed cells were thenincubated for 10 minutes with 100 μL of 0.4% sulforhodamine B (SRB) in1% acetic acid. The supernatant was removed, and the plates were washedfive times with 1% acetic acid and air dried. The bound stain was thendissolved in 10 mM trizma base and the absorbance was read using anautomated plate reader at 515 nm.

Suspension cells were treated in the same fashion with the exceptionthat the cells were fixed once they had settled at the bottom of thewells using 50 μL of 80% TCA. The absorbance of each well was thenconverted to growth percent using equation 1 (below) where T_(i) is theaverage absorbance of the samples treated with a certain concentrationof the compound of this invention, T_(z) represents the averageabsorbance of the samples fixed at the time the compound of interest wasadded to the experimental groups, and C is the average absorbance of themedia treated control.

$\begin{matrix}{{{Growth}\mspace{14mu} {Percent}} = \left\{ \begin{matrix}{{\frac{T_{i} - T_{z}}{C - T_{z}}*100},} & {{{for}\mspace{14mu} T_{i}} \geq T_{z}} \\{{\frac{T_{i} - T_{z}}{T_{z}}*100},} & {{{for}\mspace{14mu} T_{i}} < T_{z}}\end{matrix} \right.} & (1)\end{matrix}$

Cell Cycle Analysis

The effect of the deacetylnemorone on the cell cycle of SKMEL5 melanomacells was determined using a PI staining assay. SKMEL5 cells weremaintained in RPMI 1640 media supplemented with 10% FBS and 2 mML-glutamine and incubated at 37° C. and 5% CO₂. The cells were seeded in6 well plates at a density of 300,000 cells per well. After 24 hours,the media was removed and replaced with either fresh media (control) orwith media supplemented with 17.3 μM of the deacetylnemorone of FIG. 1.The cells were incubated with this treatment for 72 hours total. At 6,12, 24, 48, and 72 hours, the cells from two wells each of the controlof the treated group were trypsinized, collected in centrifuge tubes,and centrifuged at 2500 rpm for 5 minutes. The supernatant was removed,and the pellet was washed with PBS and centrifuged 2 times. The pelletwas then suspended in 500 μL PBS and added 3 mL of 70% ethanol dropwisewhile vortexing. This mixture was then incubated at 4° C. for 24 hours.Next, the mixture was centrifuged, the supernatant was removed and thepellet was suspended in PBS and stored at 4° C. until all the sampleswere collected. Once all of the samples were prepared, the cells wereagain centrifuged, the supernatant was discarded, and the pellet wassuspended in 500 μL of FxCycle PI/RNase staining solution and incubatedat 4° C. for 24 hours before analyzing the cells using a BDLSR II flowcytometer. The cells were gated to remove cell debris and any eventscontaining multiple cells.

In Vitro Invasion Assay

Cell migration of MDA-MB-231 breast cancer cells was assessed throughmaking a cell-free gap with a Culture Insert 2 well 24 (Ibidi),consisting of two wells that were separated by a wall. A total of 70 μlof cell suspension comprising of 35×10³ cells was added to each well.Cells were given 24 hours to attach and reach confluency. Cultureinserts were then removed and cell debris was washed with PBS. Freshculture media supplemented with different concentration of the compoundof interest was added to the wells and the cells were incubated at 37°C. and 5% CO₂ for 26 hours. Images were taken 0, 6, 20, and 26 hoursafter compound addition using a phase contrast Nikon Eclipse Ti-Einverted microscope. Quantification of invasion was performed bymeasuring the gap distance for three repetitions and using the followingformula,

$\begin{matrix}{{{Invasion}\mspace{14mu} \%} = {\frac{\left( {W_{0} - W_{n}} \right)}{W_{0}}*100\%}} & (2)\end{matrix}$

where W_(n) is the width of gap after incubation, and W₀ is the initialwidth right after forming the gap.

Tube Formation Assay

Growth factor reduced BD Matrigel (Corning), stored long-term at −20°C., was thawed on ice overnight in a 4° C. refrigerator prior to use.The following day, 50 μL of the thawed Matrigel was added to each wellof a pre-chilled 96-well plate and incubated at 37° C. and 5% CO₂ forapproximately 30 minutes to allow the matrix to form a gel. Next, 100 μLof a cell suspension containing 20,000 HUVEC cells in cell culture mediawith various concentrations of deacetylnemorone was added to each well.The control consisted of HUVEC cells suspended in endothelial growthmedia alone. After 8 hours of incubation at 37° C. and 5% CO2, thenumber of tubes formed between the endothelial cells were quantifiedusing an inverted microscope (Invitrogen EVOS FL Auto at 4×magnification).

Results

The deacetylnemorone showed significant cytotoxic properties against allcell lines screened with MTS assay at all time points as shown in FIG. 9(MG-63 cancer cells), FIG. 10 (SK-OV-3 cancer cells), FIG. 11(MDA-MB-231 cancer cells), FIG. 12 (HCT-116 cancer cells), FIG. 13 (HCT116/200 cancer cells) and FIG. 14 (A2789ADR cancer cells). The effectivedose varied from cell line to cell line, but cell death was indicatedusing the MTS assay at concentrations as low as 10 μg/mL in breast andcolon cancer.

The anti-proliferative effect of deacetylnemorone was confirmed by theNCI-60 screen as seen in FIG. 15. The growth of 55 of the 59 cell linesscreened was inhibited by the deacetylnemorone, and the average percentgrowth across all 59 cell lines had been inhibited by 47% after 48hours. The deacetylnemorone appeared to be particularly effectiveagainst melanoma, inhibiting the growth of 6 out of 9 melanoma celllines by more than 70%. Additionally, a negative percent growth wasobserved in the melanoma line SK-MEL-5. This negative percent growth ofthis melanoma line confirms that deacetylnemorone is able to induce celldeath in certain cell lines while sparing others.

A dose dependent, synergistic cytotoxic effect was observed when thedeacetylnemorone was used in combination with FdUrd as seen in FIG. 16and FIG. 17. This synergistic effect was observed using lowerconcentrations of deacetylnemorone than was capable of eliciting acytotoxic response alone. This speaks to the ability of deacetylnemoroneto induce desired levels of cell death when in combination with otherchemotherapy agents while simultaneously limiting the dosage of both theaccepted chemotherapy agent and the deacetylnemorone itself.

In addition to causing cell death in a number of cancer cell types, thedeacetylnemorone demonstrated an ability to disrupt the cell cycle asshown in FIG. 18, FIG. 19A, and FIG. 19B. SKMEL5 melanoma cellsincubated with the compound exhibited an increase in the percent ofcells in S phase after 24 hours. An increase in S phase is associatedwith inhibition of DNA or protein synthesis before mitosis can occur. Astime progressed, the cells which had been slowed through S phasegradually entered G2/M by the 72 hour time point. A low sub-G1population over the same time scale suggests that the cells are eithernot dying or dying through a non-apoptotic pathway. These resultssuggest that the deacetylnemorone is interfering with cell growth anddivision which will potentiate a cytostatic effect on rapidly growingcancerous cells. Further, higher concentration of deacetylnemorone hasthe potential to fully arrest cell cycle progression and lead to celldeath.

Deacetylnemorone has further shown an ability to inhibit the invasion ofbreast cancer cells in vitro. As shown in FIG. 20 and FIG. 21, theinvasive ability of breast cancer cells was significantly inhibited by 1or 10 μg/mL of deacetylnemorone after 26 hours. Invasion was alsosignificantly inhibited after 20 hours of incubation after just 20hours.

Deacetylnemorone was additionally able to inhibit tube formation betweenHUVEC cells at 0.1 and 10 μg/mL concentrations as shown in FIG. 22, FIG.23, and FIG. 24A-24C. The tube formation assay is used to model theformation of new blood vessels, and as a result, this findingdemonstrates the potential of deacetylnemorone to act as anantiangiogenic agent. By inducing a cytotoxic effect on cancer cells,diminishing the invasive property of the cells, and inhibitingangiogenesis, deacetylnemorone may be able to attack tumor growth, tumorformation, and metastasis through a multipronged mechanism.

While certain embodiments of the disclosed subject matter have beendescribed using specific terms, such description is for illustrativepurposes only, and it is to be understood that changes and variationsmay be made without departing from the spirit or scope of the subjectmatter.

What is claimed is:
 1. A method for inhibiting growth and development ofcancer cells, the method comprising delivering a deacetylnemoroneabietane diterpenoid to an area comprising cancer cells.
 2. The methodof claim 1, wherein the deacetylnemorone abietane diterpenoid has astructure as follows:

or a tautomer there of in which R₁, R₂, and R₃ are independentlyselected from —H, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ alkenyl, C₁₋₁₀alkenoxy, —OH, —OAc, —CHO, -Ph, —OC₆H₅, —OC₆H₄OH, —COC₆H₅, —OCONH₂,—OCONHCH₃, —OCOC₆H₄NH₂, —NH₂, or ═O.
 3. The method of claim 1, whereinthe deacetylnemorone is delivered to the cancer cells such that thedeacetylnemorone contacts the cancer cells at a concentration of about10 millimolar or greater.
 4. The method of claim 1, wherein thedeacetylnemorone is delivered to the cancer cells such that thedeacetylnemorone contacts the cancer cells at a concentration of fromabout 10 millimolar to about 50 millimolar.
 5. The method of claim 1,wherein the deacetylnemorone is delivered to the area in multiple doses.6. The method of claim 1, wherein the cancer cells comprise breastcancer cells, bladder cancer cells, Kaposi's sarcoma cells, lymphomacells, ovarian cancer cells, prostate cancer cells, central nervoussystem cancer cells, renal cancer cells, melanoma cells, colon cancercells, non-small cell lung cancer cells, or leukemia cells.
 7. Themethod of claim 1, the method comprising delivering the deacetylnemoroneto the cancer cells in conjunction with a second bioactive agent.
 8. Themethod of claim 7, the method comprising delivering the deacetylnemoroneand the second bioactive agent to the cancer cells together in a singlecomposition.
 9. The method of claim 7, the method comprising deliveringthe deacetylnemorone separately from the delivery of the secondbioactive agent to the area.
 10. The method of claim 7, wherein thesecond bioactive agent comprises a chemotherapy agent.
 11. The method ofclaim 10, wherein the chemotherapy agent comprises5-fluoro-2′-deoxyuridine.
 12. The method of claim 1, wherein the cancercells are resistant to one or more chemotherapy agents.
 13. A method forinhibiting angiogenesis, the method comprising delivering adeacetylnemorone abietane diterpenoid to an area comprising endothelialcells.
 14. The method of claim 13, wherein the deacetylnemorone abietanediterpenoid has a structure as follows:

or a tautomer there of in which R₁, R₂, and R₃ are independentlyselected from —H, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ alkenyl, C₁₋₁₀alkenoxy, —OH, —OAc, —CHO, -Ph, —OC₆H₅, —OC₆H₄OH, —COC₆H₅, —OCONH₂,—OCONHCH₃, —OCOC₆H₄NH₂, —NH₂, or ═O.
 15. The method of claim 13, whereinthe deacetylnemorone contacts the epithelial cells at a concentration offrom about 0.1 μg/m L to about 10 μg/m L.
 16. The method of claim 13,the area further comprising cancer cells.
 17. The method of claim 13,the method comprising delivering the deacetylnemorone to the area inconjunction with a second bioactive agent.
 18. A composition comprisinga deacetylnemorone abietane diterpenoid and a pharmaceuticallycompatible carrier, the deacetylnemorone having the following structure:

or a tautomer there of in which R₁, R₂, and R₃ are independentlyselected from —H, C₁₋₁₀ alkyl, C₁₋₁₀ alkoxy, C₁₋₁₀ alkenyl, C₁₋₁₀alkenoxy, —OH, —OAc, —CHO, -Ph, —OC₆H₅, —OC₆H₄OH, —COC₆H₅, —OCONH₂,—OCONHCH₃, —OCOC₆H₄NH₂, —NH₂, or ═O.
 19. The composition of claim 18,further comprising a second bioactive agent.
 20. The composition ofclaim 19, wherein the second bioactive agent is a chemotherapy agent.21. The composition of claim 20, wherein the chemotherapy agent exhibitsactivity against breast cancer cells, bladder cancer cells, Kaposi'ssarcoma cells, lymphoma cells, or leukemia cells.
 22. The composition ofclaim 18, wherein the composition is configured for in vivo delivery.